The long term objective of this grant proposal is to understand how the ribosome functions at the atomic level during protein synthesis. Our approach includes developing genetic and biochemical systems in the extreme thermophile Thermus thermophilus to construct and isolate mutants in rRNA, ribosomal proteins and translation factors. Our first aim is to characterize wild-type T. thermophilus ribosomes in solution by structure probing and determine modified residues by mass spectrometry. This will serve as the baseline for subsequent analysis of mutants in rRNA and ribosomal proteins. Selected mutants will be identified for crystallization attempts. A second aim is to investigate the nature of interactions between the ribosome and elongation factors EF-Tu and EF-G during accommodation and translocation. Selected functional sites in both subunits will be probed. A model for conformational change during translocation will be evaluated as will the mechanism of translocation inhibition by tuberactinomycin antibiotics. A third aim will probe our collection of mutants in the peptidyltransferase active site of 23S rRNA of T. thermophilus to characterize functions in addition to peptide bond formation associated with this region, including translocation, decoding and peptide release. Fast kinetic experiments will be used to evaluate the mutants and test a model of accommodation, and the peptide antibiotic negamycin will be examined for its role in termination. These experiments should provide fundamental insights into ribosome structure-function relationships, different conformational states of the ribosome during translation and the mode of action of and resistance to clinically useful antibiotics.